Genome size evolution is associated with climate seasonality and glucosinolates, but not life history, soil nutrients or range size, across a clade of mustards.

BACKGROUND AND AIMS: We investigate patterns of evolution of genome size across a morphologically and ecologically diverse clade of Brassicaceae, in relation to ecological and life history traits. While numerous hypotheses have been put forward regarding autecological and environmental factors that could favour small vs. large genomes, a challenge in understanding genome size evolution in plants is that many hypothesized selective agents are intercorrelated. METHODS: We contribute genome size estimates for 47 species of Streptanthus Nutt. and close relatives, and take advantage of many data collections for this group to assemble data on climate, life history, soil affinity and composition, geographic range and plant secondary chemistry to identify simultaneous correlates of variation in genome size in an evolutionary framework. We assess models of evolution across clades and use phylogenetically informed analyses as well as model selection and information criteria approaches to identify variables that can best explain genome size variation in this clade. KEY RESULTS: We find differences in genome size and heterogeneity in its rate of evolution across subclades of Streptanthus and close relatives. We show that clade-wide genome size is positively associated with climate seasonality and glucosinolate compounds. Model selection and information criteria approaches identify a best model that includes temperature seasonality and fraction of aliphatic glucosinolates, suggesting a possible role for genome size in climatic adaptation or a role for biotic interactions in shaping the evolution of genome size. We find no evidence supporting hypotheses of life history, range size or soil nutrients as forces shaping genome size in this system. CONCLUSIONS: Our findings suggest climate seasonality and biotic interactions as potential forces shaping the evolution of genome size and highlight the importance of evaluating multiple factors in the context of phylogeny to understand the effect of possible selective agents on genome size.

Construction of a Shape-Diverse Fragment Set: Design, Synthesis and Screen against Aurora-A Kinase.

Historically, chemists have explored chemical space in a highly uneven and unsystematic manner. As an example, the shape diversity of existing fragment sets does not generally reflect that of all theoretically possible fragments. To assess experimentally the added value of increased three dimensionality, a shape-diverse fragment set was designed and collated. The set was assembled by both using commercially available fragments and harnessing unified synthetic approaches to sp3 -rich molecular scaffolds. The resulting set of 80 fragments was highly three-dimensional, and its shape diversity was significantly enriched by twenty synthesised fragments. The fragment set was screened by high-throughput protein crystallography against Aurora-A kinase, revealing four hits that targeted the binding site of allosteric regulators. In the longer term, it is envisaged that the fragment set could be screened against a range of functionally diverse proteins, allowing the added value of more shape-diverse screening collections to be more fully assessed.

Convergent evolution in floral morphology in a plant ring species, the Caribbean Euphorbia tithymaloides.

PREMISE: Ring species have long fascinated evolutionary biologists for their potential insights into lineage divergence and speciation across space. Few studies have investigated the potential for convergent or parallel evolution along the diverging fronts of ring species. We investigated a potential case of parallel floral variation in the Caribbean spurge Euphorbia tithymaloides, the only plant system with molecular support as a ring species. The terminal populations of each front, despite being the most divergent, exhibit such similar floral traits that they were originally considered each other's closest relative. METHODS: We evaluated convergence in floral and leaf traits in relation to geography across 95 populations spanning the distribution of E. tithymaloides. We also reanalyzed available genetic data (from previous phylogenetic analyses) in an explicitly spatial framework. RESULTS: Floral morphology appears to have shifted in a convergent fashion along both geographic fronts of E. tithymaloides, resulting in shorter and more compact inflorescences in Antillean populations compared to the typical elongate "slipper-like" cyathia characteristic of the area of origin. Patterns of spatial genetic variation were more consistent with a two-fronted invasion of the Caribbean than with a simpler model of isolation-by-distance. CONCLUSIONS: Floral divergence in E. tithymaloides is consistent with convergent evolution along the two fronts of a ring species. We outline several (not mutually exclusive) mechanisms that could be driving patterns in morphology, including shifts toward generalized pollination with reduced reliance on hummingbirds, shifts in floral structure closely matching available hummingbird bill traits, and shifts toward increased selfing.

An Integrated Quantitative Index for Measuring Chronic Multisite Pain: The Multiple Areas of Pain (MAP) Study.

OBJECTIVE: Despite the high prevalence of chronic multisite pain, there is little consensus on methods to characterize it. Commonly used assessments report only one dimension of pain, that is, intensity, thus ignoring the spatial aspect of pain. We developed a novel pain quantification index, the Integrated Pain Quantification Index (IPQI), on a scale of 0 to 1 that integrates multiple distinct pain measures into a single value, thus representing multidimensional pain information with a single value. DESIGN: Single-visit, noninterventional, epidemiological study. SETTING: Fourteen outpatient multidisciplinary pain management programs. PATIENTS: Patients with chronic pain of the trunk and/or limbs for at least six months with average overall pain intensity of at least 5 on the numeric rating scale. METHODS: Development of IPQI was performed in a large population (N = 810) of chronic pain patients from the Multiple Areas of Pain (MAP) study. RESULTS: Prevalence of two or more noncontiguous painful areas was at 88.3% (95% confidence interval [CI] = 0.86-0.90), with a mean of 6.3 areas (SD = 5.57 areas). Prevalence of more than 10% body area in pain was at 52.8% (95% CI = 0.49-0.56), with a mean at 16.1% (17.16%). On average, IPQI values were near the middle of the scale, with mean and median IPQI at 0.52 (SD = 0.13) and 0.55, respectively. The IPQI was generalizable and clinically relevant across all domains recommended by the Initiative on Methods, Measurement, and Pain Assessment in Clinical Trials. CONCLUSIONS: IPQI provided a single pain score for representing complex, multidimensional pain information on one scale and has implications for comparing pain populations across longitudinal clinical trials.

Twentieth-century shifts in forest structure in California: Denser forests, smaller trees, and increased dominance of oaks.

We document changes in forest structure between historical (1930s) and contemporary (2000s) surveys of California vegetation through comparisons of tree abundance and size across the state and within several ecoregions. Across California, tree density in forested regions increased by 30% between the two time periods, whereas forest biomass in the same regions declined, as indicated by a 19% reduction in basal area. These changes reflect a demographic shift in forest structure: larger trees (>61 cm diameter at breast height) have declined, whereas smaller trees (<30 cm) have increased. Large tree declines were found in all surveyed regions of California, whereas small tree increases were found in every region except the south and central coast. Large tree declines were more severe in areas experiencing greater increases in climatic water deficit since the 1930s, based on a hydrologic model of water balance for historical climates through the 20th century. Forest composition in California in the last century has also shifted toward increased dominance by oaks relative to pines, a pattern consistent with warming and increased water stress, and also with paleohistoric shifts in vegetation in California over the last 150,000 y.

7-(Pyrazol-4-yl)-3H-imidazo[4,5-b]pyridine-based derivatives for kinase inhibition: Co-crystallisation studies with Aurora-A reveal distinct differences in the orientation of the pyrazole N1-substituent.

Introduction of a 1-benzyl-1H-pyrazol-4-yl moiety at C7 of the imidazo[4,5-b]pyridine scaffold provided 7a which inhibited a range of kinases including Aurora-A. Modification of the benzyl group in 7a, and subsequent co-crystallisation of the resulting analogues with Aurora-A indicated distinct differences in binding mode dependent upon the pyrazole N-substituent. Compounds 7a and 14d interact with the P-loop whereas 14a and 14b engage with Thr217 in the post-hinge region. These crystallographic insights provide options for the design of compounds interacting with the DFG motif or with Thr217.

Mutualist-mediated effects on species' range limits across large geographic scales.

Understanding the processes determining species range limits is central to predicting species distributions under climate change. Projected future ranges are extrapolated from distribution models based on climate layers, and few models incorporate the effects of biotic interactions on species' distributions. Here, we show that a positive species interaction ameliorates abiotic stress, and has a profound effect on a species' range limits. Combining field surveys of 92 populations, 10 common garden experiments throughout the range, species distribution models and greenhouse experiments, we show that mutualistic fungal endophytes ameliorate drought stress and broaden the geographic range of their native grass host Bromus laevipes by thousands of square kilometres (~ 20% larger) into drier habitats. Range differentiation between fungal-associated and fungal-free grasses was comparable to species-level range divergence of congeners, indicating large impacts on range limits. Positive biotic interactions may be underappreciated in determining species' ranges and species' responses to future climates across large geographic scales.

Cytogeography and genome size variation in the Claytonia perfoliata (Portulacaceae) polyploid complex.

BACKGROUND AND AIMS: Genome duplication is a central process in plant evolution and contributes to patterns of variation in genome size within and among lineages. Studies that combine cytogeography with genome size measurements contribute to our basic knowledge of cytotype distributions and their associations with variation in genome size. METHODS: Ploidy and genome size were assessed with direct chromosome counts and flow cytometry for 78 populations within the Claytonia perfoliata complex, comprised of three diploid taxa with numerous polyploids that range to the decaploid level. The relationship between genome size and temperature and precipitation was investigated within and across cytotypes to test for associations between environmental factors and nuclear DNA content. KEY RESULTS: A euploid series (n = 6) of diploids to octoploids was documented through chromosome counts, and decaploids were suggested by flow cytometry. Increased variation in genome size among populations was found at higher ploidy levels, potentially associated with differential contributions of diploid parental genomes, variation in rates of genomic loss or gain, or undetected hybridization. Several accessions were detected with atypical genome sizes, including a diploid population of C. parviflora ssp. grandiflora with an 18 % smaller genome than typical, and hexaploids of C. perfoliata and C. parviflora with genomes 30 % larger than typical. There was a slight but significant association of larger genome sizes with colder winter temperature across the C. perfoliata complex as a whole, and a strong association between lower winter temperatures and large genome size for tetraploid C. parviflora. CONCLUSIONS: The C. perfoliata complex is characterized by polyploids ranging from tetraploid to decaploid, with large magnitude variation in genome size at higher ploidy levels, associated in part with environmental variation in temperature.

Polyploidy associated with altered and broader ecological niches in the Claytonia perfoliata (Portulacaceae) species complex.

PREMISE OF THE STUDY: Polyploids are often hypothesized to have distinct and broader niches than their diploid progenitors. Differences in geographic distributions between diploid and polyploids are frequently used to infer niche differentiation and increased breadth, but they are seldom used to test these hypotheses explicitly. METHODS: Niche overlap and breadth were compared for diploids, tetraploids, and hexaploids of three taxa in the Claytonia perfoliata complex (C. parviflora, C. perfoliata, and C. rubra) with the use of species distribution models. Resampling and randomization approaches were used to test hypotheses of niche differentiation, breadth, and conservatism. KEY RESULTS: Niche differentiation was detected between polyploid and diploid cytotypes assigned to the same taxon (e.g., C. parviflora 2× vs. 4×) but not between hexaploids and tetraploids within a taxon (e.g., C. parviflora 4× vs. 6×). Individual hexaploid cytotypes had broader ecological niches than individual diploid cytotypes. However, as a group the three hexaploid taxa did not exceed the combined niche breadth of the three diploids, suggesting that polyploidy does not result in transgressive niche breadth for this group. Niche overlap was lowest among diploids and was highest among the three hexaploid cytotypes, consistent with introgression associated with polyploidy resulting in greater ecological similarity. Although cytotypes possessed nonidentical niches, after accounting for environmental differences among ranges, cytotypes were more similar than expected, suggesting niche conservatism and similar responses to environmental characteristics. CONCLUSIONS: These results suggest that polyploids occupy distinct and broader niches relative to diploids but that cytotypes also share fundamentally similar responses to environmental variation across ploidy levels.

Type II Kinase Inhibitors Targeting Cys-Gatekeeper Kinases Display Orthogonality with Wild Type and Ala/Gly-Gatekeeper Kinases.

Analogue-sensitive (AS) kinases contain large to small mutations in the gatekeeper position rendering them susceptible to inhibition with bulky analogues of pyrazolopyrimidine-based Src kinase inhibitors (e.g., PP1). This "bump-hole" method has been utilized for at least 85 of ∼520 kinases, but many kinases are intolerant to this approach. To expand the scope of AS kinase technology, we designed type II kinase inhibitors, ASDO2/6 (analogue-sensitive "DFG-out" kinase inhibitors 2 and 6), that target the "DFG-out" conformation of Cys-gatekeeper kinases with submicromolar potency. We validated this system in vitro against Greatwall kinase (GWL), Aurora-A kinase, and cyclin-dependent kinase-1 and in cells using M110C-GWL-expressing mouse embryonic fibroblasts. These Cys-gatekeeper kinases were sensitive to ASDO2/6 inhibition but not AS kinase inhibitor 3MB-PP1 and vice versa. These compounds, with AS kinase inhibitors, have the potential to inhibit multiple AS kinases independently with applications in systems level and translational kinase research as well as the rational design of type II kinase inhibitors targeting endogenous kinases.

An experimental test of local adaptation among cytotypes within a polyploid complex.

The geographic distributions of polyploids suggest they can have distinct and sometimes broader niches compared to diploids. However, relatively few field experiments have investigated whether range differences are associated with local adaptation or reflect other processes, such as dispersal limitation. In three years of transplants across the elevational ranges of five cytotypes in the Claytonia perfoliata complex, we found evidence for local adaptation. In at least one study year germination was higher within the natural range for each cytotype, and four of the five cytotypes attained larger biomass within their natural range. Fitness within and beyond range varied across years, with two instances of cytotypes showing higher fitness beyond the range, highlighting a potential role of temporal variability in cytotype differentiation. Polyploids as a group did not outperform diploids, but the cytotype with highest fitness across environments was a hexaploid reported to be invasive. Our results suggest that differences in geographic ranges within the C. perfoliata complex reflect local adaptation of cytotypes. Although we did not find a general polyploid advantage, our findings support the idea that occasional polyploid cytotypes exhibit high fitness relative to other cytotypes, and contribute to growing evidence supporting ecological differentiation of cytotypes within polyploid complexes.

Switching Aurora-A kinase on and off at an allosteric site.

Protein kinases are central players in the regulation of cell cycle and signalling pathways. Their catalytic activities are strictly regulated through post-translational modifications and protein-protein interactions that control switching between inactive and active states. These states have been studied extensively using protein crystallography, although the dynamic nature of protein kinases makes it difficult to capture all relevant states. Here, we describe two recent structures of Aurora-A kinase that trap its active and inactive states. In both cases, Aurora-A is trapped through interaction with a synthetic protein, either a single-domain antibody that inhibits the kinase or a hydrocarbon-stapled peptide that activates the kinase. These structures show how the distinct synthetic proteins target the same allosteric pocket with opposing effects on activity. These studies pave the way for the development of tools to probe these allosteric mechanisms in cells.

Characterization of Three Druggable Hot-Spots in the Aurora-A/TPX2 Interaction Using Biochemical, Biophysical, and Fragment-Based Approaches.

The mitotic kinase Aurora-A and its partner protein TPX2 (Targeting Protein for Xenopus kinesin-like protein 2) are overexpressed in cancers, and it has been proposed that they work together as an oncogenic holoenzyme. TPX2 is responsible for activating Aurora-A during mitosis, ensuring proper cell division. Disruption of the interface with TPX2 is therefore a potential target for novel anticancer drugs that exploit the increased sensitivity of cancer cells to mitotic stress. Here, we investigate the interface using coprecipitation assays and isothermal titration calorimetry to quantify the energetic contribution of individual residues of TPX2. Residues Tyr8, Tyr10, Phe16, and Trp34 of TPX2 are shown to be crucial for robust complex formation, suggesting that the interaction could be abrogated through blocking any of the three pockets on Aurora-A that complement these residues. Phosphorylation of Aurora-A on Thr288 is also necessary for high-affinity binding, and here we identify arginine residues that communicate the phosphorylation of Thr288 to the TPX2 binding site. With these findings in mind, we conducted a high-throughput X-ray crystallography-based screen of 1255 fragments against Aurora-A and identified 59 hits. Over three-quarters of these hits bound to the pockets described above, both validating our identification of hotspots and demonstrating the druggability of this protein-protein interaction. Our study exemplifies the potential of high-throughput crystallography facilities such as XChem to aid drug discovery. These results will accelerate the development of chemical inhibitors of the Aurora-A/TPX2 interaction.

A TPX2 Proteomimetic Has Enhanced Affinity for Aurora-A Due to Hydrocarbon Stapling of a Helix.

Inhibition of protein kinases using ATP-competitive compounds is an important strategy in drug discovery. In contrast, the allosteric regulation of kinases through the disruption of protein-protein interactions has not been widely adopted, despite the potential for selective targeting. Aurora-A kinase regulates mitotic entry and mitotic spindle assembly and is a promising target for anticancer therapy. The microtubule-associated protein TPX2 activates Aurora-A through binding to two sites. Aurora-A recognition is mediated by two motifs within the first 43 residues of TPX2, connected by a flexible linker. To characterize the contributions of these three structural elements, we prepared a series of TPX2 proteomimetics and investigated their binding affinity for Aurora-A using isothermal titration calorimetry. A novel stapled TPX2 peptide was developed that has improved binding affinity for Aurora-A and mimics the function of TPX2 in activating Aurora-A's autophosphorylation. We conclude that the helical region of TPX2 folds upon binding Aurora-A, and that stabilization of this helix does not compromise Aurora-A activation. This study demonstrates that the preparation of these proteomimetics using modern synthesis methods is feasible and their biochemical evaluation demonstrates the power of proteomimetics as tool compounds for investigating PPIs involving intrinsically disordered regions of proteins.